Bordetella pertussis, the causative agent of whooping cough, produces a number of factors which contribute to its pathogenic potential. Expression of the genes encoding these factors is under the overall control of the bvg locus, which encodes a two component signal transduction (response regulator) system consisting of an environmental sensor protein and a DNA- binding transcriptional activator protein. However, evidence suggests that the bvg system directly activates only some of the genes in the bvg regulon (such as bvg and fha), whereas others (such as ptx and cya) apparently require additional factors for their activation. Two mutant strains of B. pertussis have greatly decreased levels of transcription of the ptx and cya genes, while transcription of other genes of the bvg regulon is normal, a phenotype which suggests a defect in an additional factor specifically for ptx and cya promoter activation. This proposal will focus on the identification of such additional regulatory factors in the bvg regulon, particularly those which activate the ptx (and cya) promoters. Random and targeted transposon mutagenesis will be performed to obtain mutants with phenotypes suggesting defects in these regulatory factors. Cloning of the genes encoding ptx promoter activating factors from a library of B. pertussis DNA will be attempted by two methods: activation of a ptx promoter-reporter gene fusion in Escherichia coli and probing for ptx promoter sequence-specific binding proteins in a lambdalgt11 expression library. The mutant strains with decreased ptx and cya transcription will be analyzed further to identify the specific defect involved, and the wild type gene corresponding to the mutant gene in these strains will be cloned by complementation of the mutants. Also, the promoters of the other known genes of the bvg regulon will be analyzed to determine whether they are directly activated by bvg or require additional regulatory factors, and identification and cloning of these factors will be attempted using the same methods as for the ptx promoter activating factors. An understanding of the regulatory events involved in expression of virulence factors in B. pertussis will provide further insight into the infection and disease process of this pathogen and will be useful for development of improved vaccines to prevent the disease.